KR20190022202A - Direct conversion method from somatic cells to induced cholangiocyte stem cells - Google Patents

Abstract

The present invention relates to a transdifferentiation method from somatic cells to induced cholangiocyte stem cells. The method of transdifferentiation into the induced cholangiocyte stem cells of the present invention will be useful for the development of a cell therapeutic agent using cholangiocyte stem cells, modeling of diseases related to cholangiocytes, and studies on a mechanism of development and differentiation of cholangiocytes.

Description

{Direct conversion method from somatic cells to induced cholangiocyte stem cells}

The present invention relates to a method of crossing differentiation from somatic cells into inducible biliary stromal cells and induction biliary stromal cells produced thereby.

Liver transplantation has been the only treatment for long-term treatment of chronic liver disease. However, the treatment method through liver transplantation is not as effective as many demands, but as a result of severe shortage of liver donors, And immune rejection after transplantation.

However, recently, a method to fundamentally treat chronic liver disease through the cellular therapeutic approach using adult hepatocyte isolated from the adult has begun to be popularized. Nevertheless, it has been recognized that cell therapy methods that directly transplant adult stem cells have great difficulties in utilization due to the limit of in vitro survival rate and proliferative capacity due to hepatocyte characteristics (Hay et al. (2008) Stem Cells 26, 894-902; He, J. et al. (2014) Gastroenterology 146, 789-800 e 788; Si-Tayeb et al., (2010) Hepatology 51, 297-305).

As an alternative to this, cell therapy using hepatocyte-like cells derived from pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) has been developed. However, (Tang, C. et al., Nat. Biotechnol., 29, 829-834), because the possibility of tumor formation due to tumor growth is not fundamentally excluded.

To overcome these limitations, methods for producing and transplanting induced hepatocytes (iHeps) have been studied using limited direct conversion technology, which can displace the possibility of tumor formation from 2012 onwards.

Recently, it has been shown that cells such as non-hepatocytes, ie cholangiocytes, belonging to a relatively small part of the liver are very important for hepatic regeneration, so that they can produce both hepatocytes and bile duct cells from somatic cells Studies are underway to produce induced hepatic stem-like cells (iHepSCs) with differentiation potential.

Meanwhile, a cross-differentiation study (Yu B et al., Cell Stem Cell 2013; 13: 328-340) has been published from somatic cells to inducible stem cells capable of differentiating into hepatocytes and biliary duct cells, However, the efficiency of differentiation into restrictive bile duct cells acted as a stumbling block.

Recently, a method for producing biliary stromal cells and biliary duct cells using human pluripotent stem cells has been developed (Ogawa M et al., Nat Biotechnol 2015; 33: 853-861; Sampaziotis F et al., Nat Biotechnol 2015; 33: 845-852), the possibility of tumor formation due to the activation of tumor cell genes was not able to be excluded from the technical point of view, thus showing a limit in clinical application.

Therefore, the present inventors propose a method for improving the low cross-over differentiation efficiency from somatic cells to biliary stromal cells.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Reference Patent Document

Korean Patent No. 10-1587231 Compositions and methods for promoting direct crossing differentiation from somatic cells to hepatocytes

Method for enhancing direct crossing differentiation efficiency from human somatic cell to hepatocyte using small molecule compound

Korea Patent No. 10-1639595 Compositions and methods for promoting direct crossing differentiation from somatic cells to hepatocytes

The present inventors have tried to develop a method for improving the cross-differentiation efficiency of induced cholangiocyte stem cells in somatic cells. As a result, the inventors of the present invention developed a combination of (i) strictly selecting transcription factors necessary for hepatic development and liver regeneration and eventually developed a combination of 1a3 (Hnf1α, Foxa3), and introduced the gene of the combination into somatic cells to induce inducible stem cell (step 1), and (ii) followed by continuous in vitro culture to induce stem cell differentiation into cholangiocyte stem cells (CPC), which is a pre-cholangiocyte stem cell (step 2).

Accordingly, an object of the present invention is to provide a method of crossing differentiation into induced cholangiocyte stem cells.

Another object of the present invention is to provide a composition for cross-differentiation into induced cholangiocyte stem cells.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a method of crossing differentiation into induced hepatic stem-like cells in somatic cells comprising the step of introducing Hnf1? And Foxa3 genes into somatic cells of mammals isolated in vitro to provide.

According to the present invention, the Hnf1α (HNF1 homeobox A) gene comprises a nucleotide sequence represented by SEQ ID No. 1, and the Foxa3 (forkhead box A3) gene comprises a nucleotide sequence represented by SEQ ID No. 2 . According to an embodiment of the present invention, the Hnf1? Gene is composed of a nucleotide sequence represented by SEQ ID No. 1 and the Foxa3 gene is composed of a nucleotide sequence represented by SEQ ID No. 2.

It will be apparent to those skilled in the art that the first and second sequences of the Sequence Listing in the present invention are not limited to the nucleotide sequences described in the attached Sequence Listing.

Variations in nucleotides do not cause changes in the protein. Such nucleic acids include functionally equivalent codons or codons that encode the same amino acid (e.g., by codon degeneration, six codons for arginine or serine), or codons that encode biologically equivalent amino acids ≪ / RTI >

Considering the mutation having the above-mentioned biological equivalent activity, the nucleic acid molecule used in the present invention is interpreted to include a sequence showing substantial identity with the sequence described in the sequence listing. The above-mentioned substantial identity is determined by aligning the sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. Homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology.

Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are described by Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is accessible from National Center for Biological Information (NBCI) It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. BLSAT is available at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

On the other hand, the Hnf1? Gene or Foxa3 gene sequence may comprise the CDS portion of Sequence Listing first and second sequences, respectively.

According to the present invention, the gene sequence of the present invention (or the nucleotide sequence for expression of the gene) is contained in a suitable gene delivery system. According to one embodiment of the present invention, the gene carrier may be a virus, a plasmid, a liposome, or a niozyme in the gene introduction, but is not limited thereto.

As used herein, the term " gene transporter " means an agent for introducing and expressing a desired target gene into a target cell. The ideal gene transporter is harmless to the human body, is easy to mass-produce, and must be able to efficiently transfer the gene.

As used herein, the term " gene transfer " means that a gene is carried into a cell and has the same meaning as transduction of a gene into a cell. At the tissue level, the term gene transfer has the same meaning as the spread of a gene. Accordingly, the gene carrier of the present invention can be described as a gene penetration system and a gene diffusion system.

To prepare the gene delivery vehicle of the present invention, the nucleotide sequence of the present invention is preferably present in a suitable expression construct. In such expression constructs, the nucleotide sequence of the invention is preferably operatively linked to a promoter. As used herein, the term " operably linked " means a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter, signal sequence, or transcription factor binding site) and another nucleic acid sequence, The regulatory sequence will regulate transcription and / or translation of the other nucleic acid sequences. In the present invention, the promoter bound to the nucleotide sequence of the present invention is preferably capable of regulating the transcription of the LAMTOR3 gene by working in an animal cell, more preferably a mammalian cell, and comprises a promoter derived from a mammalian virus and A promoter derived from the genome of a mammalian cell, such as CMV (mammalian cytomegalovirus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter Promoter of the human IL-2 gene, the promoter of the human IFN gene, the promoter of the human IL-4 gene, the promoter of the human lymphotoxin gene and the promoter of the human GM-CSF gene, U6 Promoters, but are not limited thereto.

The gene carrier of the present invention can be produced in various forms including (i) naked recombinant DNA molecules, (ii) plasmids, (iii) viral vectors, and (iv) the naked recombinant DNA molecules or plasmids In the form of a liposome or a niozyme.

Hnf1? And Foxa3 gene nucleotide sequences can be applied to all gene delivery systems used in conventional gene therapy and are preferably plasmids, adenoviruses (Lockett LJ, et al., Clin. Cancer Res. 3: 2075-2080 ), Adeno-associated viruses (AAV, Lashford LS., Et al., Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), retrovirus (Gunzburg WH, et al., Retroviral (Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), lentivirus (Wang G. et al., J. Clin. Invest. 104 (11): R55-62 Viruses (Puhlmann M. et al., Human Gene Therapy 10: 649-657 (1999)), viruses (Chamber R., et al., Proc. Natl. Act. Sci. USA 92: 1411-1415 ), Liposomes (Methosin in Molecular Biology, Vol. 199, SC Basu and M. Basu (Eds.), Human Press 2002) or niosomes. According to one embodiment of the present invention, the gene transporter can be produced by applying the gene sequence or the nucleotide molecule of the present invention to a retrovirus.

In the present invention, when the gene carrier is prepared based on a viral vector, the contacting step is carried out according to a virus infection method known in the art. Infection of host cells with viral vectors is described in the above cited documents.

22: 479 (1980); and Harland and Weintraub, J. Cell Biol. 101: 1094-919), in the case where the gene carrier is a naked recombinant DNA molecule or plasmid in the present invention. 1099 (1985)), calcium phosphate precipitation method (Graham, FL et al., Virology, 52: 456 (1973), and Chen and Okayama, Mol. Cell. Biol.7: 2745-2752 6: 716-718 (1986)), liposome-mediated transfection (Eugene et al., EMBO J., 1: 841 (1982), and Tur-Kaspa et al., Mol. Cell Biol. Et al., Methods Enzymol., ≪ RTI ID = 0.0 > 149: < / RTI > (1987)), DEAE-dextran treatment (Gopal, MoI. Cell Biol., 5: 1188-1190 (1985)), and gene bend buddhite (Yang et al., Proc. Natl. , ≪ / RTI > 87: 9568-9572 (1990)).

According to the present invention, the method of the present invention can be applied to somatic cells of mammals including, but not limited to, humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys and chimpanzees no.

According to the present invention, the induction stem cell prepared by the method of the present invention is capable of differentiating into hepatocyte or bile duct cell.

According to another aspect of the present invention, the present invention provides a method of crossing differentiation from somatic cells into hepatic cells, comprising introducing Hnf1 [alpha] and Foxa3 genes into somatic cells of an ex vivo separated mammal.

According to another aspect of the present invention, there is provided a composition for crossing differentiation from somatic cells to hepatic cells, comprising a gene carrier for Hnf1? Gene and Foxa3 gene expression.

Since the method and composition for crossing differentiation into hepatocytes utilize the same gene as the above-mentioned cross-differentiation method for inducing stem cell, the common content in relation to the above method is that, in order to avoid the excessive complexity of the present invention, It is omitted.

According to another aspect of the present invention, there is provided a composition for cross-differentiation into induced hepatic stem-like cells in somatic cells, comprising a gene carrier for Hnf1? And Foxa3 gene expression.

Since the composition of the present invention uses the same gene as in the above-mentioned cross-differentiation method, common description in relation to the method is omitted in order to avoid the excessive complexity of the present specification.

According to one embodiment of the present invention, the somatic cell is a fibroblast.

According to another aspect of the present invention, there is provided a method for producing a cross-differentiating composition comprising contacting a cross-differentiating composition of the present invention described above with a live sample containing somatic cells, And provides a method of differentiation.

The term " biosample " in the present invention means an organism-derived sample. The raw sample refers to a medium comprising cells, tissues, or biological fluids of a biological source (e.g., a mammal), or somatic cells obtainable according to the present invention, Includes collected samples. According to the present invention, the biological sample is an in-vivo fluid sample including a tissue extract containing cells and cells.

According to another aspect of the present invention, the present invention provides a method of crossing differentiation into induced cholangiocyte stem cells in somatic cells comprising the steps of:

(a) introducing Hnf1 &agr; and Foxa3 genes into somatic cells of mammals isolated from in vitro, to produce inducible stem cells; And

(b) culturing the inducible stem cell of step (a) in a growth factor-containing medium to produce induced biliary stromal cells;

Hereinafter, the method of the present invention will be described in detail.

Step (a): Preparation of inducible stem cell

First, inducing liver stem cells are prepared by introducing Hnf1 alpha and Foxa3 genes into somatic cells of mammals isolated in vitro. The method of introducing the gene into somatic cells is as described above. According to one embodiment of the present invention, Foxa1, Foxa2, Foxa3, Hnf1a and Hnf1b genes known as transcription factors essential for liver development and liver regeneration are introduced into somatic cells (MEFs, mouse embryonic fibroblasts) via pMX retrovirus , And cultured in hepatic stem cell culture medium (HepCM) to establish a stem cell line.

According to one embodiment of the present invention, the composition of liver stem cell specific culture medium (HepCM) is as follows:

DMEM / F-12 supplemented with 10% FBS, 0.1 μM dexamethasone, 10 mM nicotinamide, 1% ITS (insulin-transferrin-selenium) premix and penicillin / streptomycin / glutamine and both hepatocyte growth factor and epidermal growth factor.

Step (b): Preparation of induced biliary stromal cells

Next, the induced stem cell is cultured in a medium (HepSCM) containing growth factors (for example, EGF and / or HGF) to differentiate into induced biliary stromal cells.

According to one embodiment of the present invention, the composition of the HepSCM medium is as follows: DMEM / F-12, 10% FBS (Hyclone), 0.1 μM dexamethasone (Sigma), 10 mM nicotinamide (Gibco), penicillin / streptomycin / glutamine (Invitrogen), 10 ng / ml hepatocyte growth factor (HGF), 10 ng / ml epidermal growth factor (EGF) and gelatin-coated dish.

In the medium composition, the inducible stem cells were subjected to continuous passage of about 14 to 16 times to differentiate into induced biliary stromal cells.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a method of crossing differentiation into induced cholangiocyte stem cells in somatic cells.

(b) The cross-differentiation method of the present invention to induce biliary stromal cells will be useful for the development of a cell therapeutic agent using biliary stromal cells, disease modeling related to cholangiocellular cells, and development of biliary cell differentiation and development.

Fig. 1 is a schematic diagram of an experimental process for inducing stem cell formation.
(AFP), gallbladder cells (CK7, CK19), and hepatic stem cells (HCCs), which are essential transcription factors for liver development and liver regeneration. (TROP2) specific markers were generated.
3A-3F. Optimal induction stem cell crossing differentiation combination screening. The number of AFP / CK19 positive colonies was measured when one of the transduction factors was removed, and when Foxa1, Foxa2, Foxa3, Hnf1a, and Hnf1b were removed, the induction of stem cell-specific epithelial cell colonies (Fig. 3A). Further, as a result of examining the expression pattern of hepatocyte and liver stem cell specific marker gene under the respective removal conditions, it was confirmed that when the Hnf1a and Foxa2 were removed, the overall marker expression was decreased (FIG. 3b). Based on these results, induction of stem cell induction by combination of Hnf1a, Foxa2 (hereinafter referred to as 1a2) and Hnf1a and Foxa3 (hereinafter referred to as 1a3) resulted in the expression pattern of AFP / CK19 gene and the number of positive colonies and the number of EPCAM- (FIG. 3C to FIG. 3E), and it was confirmed that a protein showing liver stem cell characteristics was remarkably expressed in the inducible stem cell produced using 1a3 (FIG. 3F). As control, hepatocytes and gallbladder cells isolated from adult rats were used, respectively.
Figures 4a-4c. Analysis of Fundamental Characteristics of Inducible Stem Cells. (Fig. 4A), qPCR (Fig. 4B), and immunofluorescence staining (Fig. 4C), which indicate the stem cell specificity of the inducible stem cell line 1-4 produced by the combination of the? It was confirmed that various hepatocytes, gallbladder cells and liver stem cell specific markers were clearly expressed.
5A-5E. Verification of hepatocyte differentiation ability of inducible stem cell. (FIG. 5A), and it was confirmed whether the induction stem cell produced by the combination of 1a3 showed differentiation characteristics of hepatocytes and gallbladder stem cells (Fig. 5B), immunofluorescence staining (Fig. 5C), PAS and ICG staining (Fig. 5D) and serum albumin secretion amount (Fig. 5E) after 7 days of induction of differentiation into hepatocytes. The expression levels of cell markers (Ggt, CK7) and liver stem cell markers (Dlk1) were markedly decreased, and at the same time, it was confirmed that the inducible liver stem cells showing distinctive functions of hepatocytes were able to differentiate into mature hepatocytes under hepatocyte differentiation conditions .
6a-6d. Verification of the ability of stem cells to induce gallbladder cell differentiation. After 7 days of induction of differentiation from the 3-dimensional medium into gallbladder cells, changes in the shape of the gallbladder structure and branch shape were observed (Fig. 6A). The characteristics of the differentiated cells were analyzed by qPCR (Fig. 6B), immunofluorescence staining (Fig. 6c), and the results were verified through MDR1 (multidrug resistance protein 1) and CFTR (cystic fibrosis transmembrane regulator) receptor activity assay (Fig. 6d) to verify the water and ion release function of the bile duct cells. To confirm the functionality of MDR1, it was confirmed through the absorption and excretion of rodamine 123 (Rho123). Forskolin (FSK) and IBMX were treated to determine the degree of swelling of the bile ducts. Compared with when there was CFTR inhibitor, And swelling by FSK and IBMX was inhibited (Fig. 6d).
Figure 7. Comparative analysis of stem cell crossing differentiation efficiency of induction liver. Cadherin or EPCAM positive cells using a flow cytometry analyzer for about 14 days after the transduction factor introduction in order to compare induction stem cell crossing differentiation efficiency through transcription factor combination (1b3) and combination (1a3) Results.
8A-8B. Comparison of basic characteristics with existing inducible stem cell lines.
Figures 9a-9c. 1b3 combination and 1a3 combination, the 1a3 combination showed a significantly higher Rho123 absorption rate (9a). In addition, the ability to differentiate gallbladder cells from existing inducible stem cell lines was compared using MDR1 receptor activity assay (9b) and qPCR technique (9c).
FIG. 10. A schematic diagram of an experimental procedure for inducing stem cell and biliary stromal cell in induction. (i) induction of somatic cells into induced stem cells using a combination of 1a3 (Hnf1α, Foxa3) transcription factors essential for liver development and liver regeneration (step 1); and (ii) And then differentiated into cholangiocyte stem cells (CPC), which is a pre-cell stage (step 2).
Figure 11. Differentiation of inducible stem cells into inducible biliary stromal cells.
12A-12D. Induction and Establishment of Stem Cells Induced by. After introduction of Foxa3 and Hnf1a into somatic cells, colonies expressing hepatocyte (AFP), biliary cell (CK7, CK19), and liver stem cell (TROP2) specific markers were generated (Fig. 12A). It was confirmed by RT-PCR, qPCR, and immunofluorescence staining that the produced stem cells of the present invention express liver stem cell specificity, and that they express various hepatocyte, biliary cell and liver stem cell-specific markers (Figs. 12B-12D).
Figures 13a-13c. Induced differentiation of stem cells into induced biliary stromal cells. As a result, the expression level of the hepatocyte markers Afp, Alb, and Hnf4a was decreased, whereas the bile duct cell markers such as CK19 and SOX9 were maintained (Figs. 13a-13b). In particular, when qPCR was used to identify various biliary cell-specific markers, the expressions of markers related to Notch signaling such as Notch2, Hes1, and Jag1 were significantly increased along with the expression of matured biliary cell-specific markers (Fig. 13c) .
14A-14D. Verification of induction of stem cell differentiation induced by Notch signaling activation. Treatment with 10 ng / ml of DAPT (N- [3,5- Difluorophenacetyl) -L-alanyl] -S-phenylglycine t-butyl ester, a γ-secretase inhibitor; Sigma) (Fig. 14 (b)). It was found that the induction of the stem cell state of the induced liver was maintained rather constantly in the case of qPCR (Fig. 14 (b)), immunofluorescence (Fig. 14C), and the MDR1 receptor activity assay (Fig. 14D).
15A-15F. Inducibility of inducible biliary stromal cells in the body. Inducible biliary stromal cells were transplanted into C57B16 / J mice to induce differentiation into the biliary duct cells, and the degree of differentiation of the transplanted induced biliary stromal cells was analyzed by immunohistochemistry. As a result, a large number of biliary structures were formed in the induction bile duct stromal cells as compared with the induction stem cell (Figs. 15A-15B). Immunostaining with bile duct cell-specific markers such as CK19 and OPN resulted in overwhelming And the induced biliary stromal cells were differentiated into functional biliary duct cells (Figs. 15C-15F).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

[Production and Characterization of Inducible Stem Cells]

Experimental Method

1. Production of inducible stem cell line

(NM_008259), Foxa2 (NM_010446), Foxa3 (NM_008260), and Hnf1a (NM_009327), which are essential transcription factors for liver development and liver regeneration via pMX retrovirus, induce stem cell crossing differentiation induction, , And Hnf1b (NM_008261) were introduced into the first 50,000 MEFs (mouse embryonic fibroblasts) and cultured in hepatic stem cell culture medium (HepCM) for 48 hours (FIG. 1). After about two weeks, it was confirmed that inducible stem cell colony with epithelial cell morphology was formed, and it was isolated to establish a stem cell line of induction liver stem cell (Du, Y. et al, (2014) Cell Stem Cell 14, 394 Nature 475, 386-389; Huang, P. et al, (2014) Cell Stem Cell 14, 370-384; Sekiya, S. et al., (2011) Nature 475, 390-393).

* Liver stem cell culture composition: DMEM / F-12 supplemented with 10% FBS (Biowest), 0.1 μM dexamethasone (Sigma), 10 mM nicotinamide (Sigma), 1% insulin-transferrin- selenium premix (Gibco) and penicillin / streptomycin / glutamine (Invitrogen), and both hepatocyte growth factor (Peprotech) and epidermal growth factor (Peprotech).

2. Optimal induction stem cell crossing differentiation combination screening

In order to select optimal cross-differentiation combinations to induce stem cell differentiation, we used 1) RT-PCR technique to express markers specific for hepatocytes (Afp, Alb), bile duct cells (CK7, CK19), liver stem cells (Epcam, Trop2) 2) Immunofluorescence staining was used to measure the number of colonies positive for AFP and CK19. 3) The number of EPCAM positive cells, a surface protein specific for liver stem cells, was measured by flow cytometry Respectively.

1) RT-PCR

RNA was extracted using Hybrid-RTM (GeneAll) for the analysis of gene expression patterns of induced stem cells, and cDNA was synthesized from total 1 μg of RNA using a reverse transcriptase kit (Applied Biosystems) In accordance with the standard method. RT-PCR was then performed using primers for each marker.

Primer sequence information for marker gene expression confirmation Gene name Genebank Number The primer sequence (F / R) Aat NM_009243 5'-CCTGCTAAACA6pCGCAGAA-3 ' 5'-TCGATGGTCAGCACAGCCTTA-3 ' Afp NM_007423 5'-CGTGATGCTTTGGGCGTTTA-3 ' 5'-GCCAAAAGGCTCACACCAAAG-3 ' Alb NM_009654 5'-AAACCTTGTCACTAGATGCAAAGACG-3 ' 5'-GGGTAGCCTGAGAAGGTTGTGG-3 ' Hnf1a NM_009327 5'-CCTGCTGCCATCCAACCATA-3 ' 5'-CCACGGTTACTGGGAAGAGGA-3 ' Hnf4a NM_008261 5'-GCCAACGATCACCAAGCAAG-3 ' 5'-TGAGGGTATGAGCCAGCAGAA-3 ' E-cadherin NM_009864 5'-TTCAAGAAGCTGGCGGACAT-3 ' 5'-CATCTCCCATGGTGCCACAC-3 ' Ttr NM_013697 5'-CCCTGCTCAGCCCATACTCCTA-3 ' 5'-TGCTTTGGCAAGATCCTGGT-3 ' CK8 NM_031170 5'-AAGCTGGTGTCCGAGTCTTCTGA-3 ' 5'-AGCTCAGGCTGGCAAGGACT-3 ' CK18 NM_010664 5'-GATCGTGGATGGCAGAGTGG-3 ' 5'-TTCCCTCCTTCTCTGCCTCAGT-3 ' Taste NM_146214 5'-AAGGCAGCCAGGAGGAGTGT-3 ' 5'-TGAGGAGGAGCAGCCCAACT-3 ' G6p NM_008061 5'-CGGATCCTGGGACAGACACA-3 ' 5'-CTTTGCATGGCGGTTGACTT-3 ' Ocln NM_008756 5'-TCGCACATCAAGAGGATGGTG -3 ' 5'-GCCTCTGGAGAGAATTGCAGAGA-3 ' Dsp NM_023842 5'-GGCTGACGGAAGAGGAAACTG -3 ' 5'-ATGGTGGCCAACAGCGACT-3 ' Col1a1 NM_007742 5'-CCCTGCCTGCTTCGTGTAAA-3 ' 5'-TCGTCTGTTTCCAGGGTTGG-3 ' Thy1 NM_009382 5'-CTTTCCCTCTCCCTCCTCCAAG-3 ' 5'-CGAGGGCTCCTGTTTCTCCTT-3 ' CK19 NM_008471 5'-CCCCAAGGCCATCTGAGCTA-3 ' 5'-GAGTAAACTTTTATCACCCCAGTCAGG-3 ' CK7 NM_033073 5'-CCTCAGGGCCTATTCCATCAA-3 ' 5'-GTCTCTCCAAGCCCACAGCTT-3 ' Ggt NM_008116 5'-CAGCTGCCTCAGACTCCAGAA-3 ' 5'-TTCCCATTCTCGTCCCTTGG-3 ' Trop2 NM_020047 5'-GAGATGAGAAGCGAACCTAGCTTGTAG-3 ' 5'-AACTTGTTTGTGGAGAGAGAAGGAAGA-3 ' Epcam NM_008532 5'-GGTGGTGTCATTAGCAGTCATCG-3 ' 5'-TGTGGATCTCACCCATCTCCTT-3 ' Dlk1 NM_010052 5'-GGATTCTGCGAGGCTGACAA-3 ' 5'-GCAGATGCACTGCCATGGTT-3 ' Notch2 NM_010928 5'-TTTGTGTCCCGCCCTTGTC-3 ' 5'-AGGGCATTTGCAGGAGAACTG-3 ' Cftr NM_021050 5'-CTGCTTGATGAGCCCAGTGC-3 ' 5'-TGAAGGGAGTCGTACTGCCAGA-3 ' Aqp1 NM_00747 5'-CGGTCATTTGGCTCTGCTGT-3 ' 5'-CACTGGTCCACACCTTCATGC-3 ' Hnf1b NM_009330 5'-GTGTCCACTGCAAGCCTGG-3 ' 5'-CCCAGAGACTGATGGTGTGGA-3 ' Jag1 NM_013822 5'-ACCTGCGTGGTCAATGGAGA-3 ' 5'-CACATTCGCACCGATACCAGTT-3 ' Sstr2 NM_009217 5'-TGCTAGAGAACACAGGGAAGCGA-3 ' 5'-TGTCGTAGTATGGCTCGGTCTGG-3 ' Hes1 NM_008235 5'-GTGAAGCACCTCCGGAACCT-3 ' 5'-CTCGTTCATGCACTCGCTGA-3 ' Mdr1 NM_011075 5'-CGAAGCAACATCAGCTCTGGA-3 ' 5'-CTTTGCTCCAGCCTGCACAC-3 ' Gpbar1 NM_174985 Gt; 5'-TGGCTCTTCCTCGAAGCACTC-3 ' Sctr NM_001012322 5'-CCATGGAGGTCCAGCTGTTCT-3 ' 5'-CGTTGCTGAAGGAGTTGCTGA-3 ' Slc4a2 NM_009207 5'-GCCTTTGCGCATGGTGGTACT-3 ' 5'-TGCCTCTGGACAGCAGCTACA-3 ' Mrp2 NM_013806 5'-ACTTTCAATGCCGGCTTCCT-3 ' 5'-TGGTCCTAGACAGCGGCAAG-3 ' Mrp3 NM_029600 5'-CCGGCTCAACACAATCATGG-3 ' 5'-TAGGCAAGTCCCGCATCCTT-3 ' Ntcp NM_001177561 5'-CAAGGCTGCTGCAACAGAAGA-3 ' 5'-ACCAGAGTTCSAGGCCATTAGGG-3 ' MaoA NM_173740 5'-GGGTTCAAGAGCCTGAGTCCA-3 ' 5'-TGATCAGCAGGAGGCCTGT-3 ' Nat2 NM_010874 5'-GGAGAATGGAACCTGGTAGGA-3 ' 5'-GACGCTGGTGATGTCTGAAGG3 ' GS NM_008131 5'-GGAGGTTATGCCTGCCCAGT-3 ' 5'-CAATGGCCTCCTCAATGCAC-3 ' Gsta4 NM_010357 5'-CAGATGGCCCCTATGTTGAG-3 ' 5'-GGGCAGAGTGGTTTTGTTGT-3 ' Sult1a1 NM_133670 5'-CCATGGCTAACTACACAACCATCC-3 ' 5'-CACAGCTAAGGAGAGGACCCTTG-3 ' Ugt1a1 NM_201645 5'-CTGGAGCTCGCGTTTGCTT-3 ' 5'-CCTTCTGCTGGAGCTGCTGA-3 ' MaoB NM_172778 5'-GTGGGCCAGGAAACCAAGAG-3 ' 5'-TTTGGCAGCCAGAACCAGAA-3 ' Mgst1 NM_019946 5'-GACCTCAGGCAGCTCATGGA-3 ' 5'-GCGTTCCACCTTCTCGTCAGT-3 ' Cyp1a1 NM_009992 5'-CCTTCCGGCATTCATCCTTC-3 ' 5'-TTTCAGGCCGGAACTCGTTT-3 ' Cyp2a5 NM_007812 5'-GCACTTCCTAGATGACAAGGGACA-3 ' 5'-CAGGCTCAACGGGACAAGAA-3 ' Cyp2d22 NM_001163472 5'-CCTCTCCTCGGCTGAGTTTCA-3 ' 5'-CGCCAGTGCATCAGGTTCA-3 ' Cyp3a11 NM_007818 5'-TTCCAGCCTTGTAAGGAAACACA-3 ' 5'-TGTACTGAATCTTTAACCAGGCATCA-3 ' Cyp3a13 NM_007819 5'-TCCTGCAGAACTTCACTGTCCA-3 ' 5'-TGGTTTCTGGTCCACAGGATACA-3 ' Cyp3a44 NM_177380 5'-TGGACCCAGGAACTGCATTG-3 ' 5'-GCATCCCGTGGCACAACTT-3 ' Gapdh NM_008084 5'-CCAATGTGTCCGTCGTGGAT-3 ' 5'-TGCCTGCTTCACCACCTTCT-3 '

Primer sequence information for foreign gene expression analysis Gene name Primer sequence exo-Foxa3 Gt; 5'-TGGTGGGCACAGGATTCACT-3 ' exo-Hnf1a Gt; 5'-AGGCCTGGATCAGCACTTCC-3 ' exo-Hnf1b Gt; 5'-AGGCCTGGATCAGCACTTCC-3 '

2) Immunofluorescence staining

For immunofluorescence staining, the cells were fixed in 4% paraformaldehyde (Sigma) at room temperature for 20 minutes and then fixed with DPBS (Biowest) containing 0.3% Triton X-100 (Sigma) and 5% FBS (Biowest) For 2 hours. The cells are then subjected to an antigen-antibody reaction in the dark at 4 ° C overnight with primary antibody. After 3 washes with DPBS, the suitably bound secondary antibody is allowed to react for 2 hours under dark conditions at room temperature. Cell nuclei were stained with Hoechst 33342 (Fluka) and observed with a fluorescence microscope or a confocal laser microscope (Fluoview FV1000-ASWv1.5; Tokyo, Japan). The primary antibodies used in the experiments were as follows: rabbit anti-E-cadherin (1: 200), mouse anti-Albumin (R & D Systems, 1: 100), mouse anti-CK7 Rabbit anti-SOX9 (Novus Biologicals, 1: 200), rabbit anti-CK19 (Abcam, 1: 250), rabbit anti-EPCAM (Santa Cruz, 1: 200), anti-TROP2 (Santa Cruz, 1: 200), goat anti-F-actin (Thermo, 1: 200), and rabbit anti-ZO-1 (Invitrogen, 1: 200).

3) Flow cytometry

For flow cytometry, the cells are fixed in 4% paraformaldehyde (Sigma) for 20 minutes and then treated with 0.3% Triton X-100 (Sigma) at room temperature for 10 minutes to allow the antibody to penetrate. Blocking is carried out with 0.3% bovine serum albumin (Sigma) for 15 minutes. Epcam or E-cadherin antibody is then reacted at 4 ° C for 30 minutes. After several washes with DPBS solution, the secondary antibody is allowed to attach for 20 minutes at 4 ° C in a dark state. Cells are washed twice in DPBS containing 0.1% Tween 20 (Sigma) and analyzed using a Calibur Flow Cytometer (Becton Dickinson). The primary antibody used for flow cytometry is as follows. rabbit anti-E-cadherin (Cell Signaling, 1: 200), goat anti-EPCAM (e-bioscience, 1:50). Experimental results were analyzed using FlowJo (Tree Star) software.

3. Characterization of inducible stem cell

Induced stem cell differentiation (1a3: Hnf1a, Foxa3) was determined by various methods. To do this, we used RT-PCR, quantitative PCR (qPCR), immunofluorescence staining, 3-D differentiation using collagen and magrigel, periodic acid staining (PAS) staining, indocyanine green (ICG) staining, ELISA , MDR1 receptor activity assay by Rho123 treatment, and CFTR receptor activity assay by forskolin treatment.

1) RT-PCR and immunofluorescence staining are as described above. In order to perform quantitative PCR analysis, qPCR was performed with primers of inducible stem cell markers using SYBR green PCR Master Mix (Applied Biosystems). Expression level was based on Gapdh gene expression and MEF (mouse embryonic fibroblast) was set as negative control. All qPCR experiments were performed using an ABI 7500 real-time PCR system (Applied Biosystems).

2) Three-dimensional differentiation culture using collagen and magrigel

In order to differentiate inducible stem cells into bile duct cells, the three-dimensional differentiation culture method described in the previous paper was used (Tanimizu et al., 2004, Journal of cell science 117, 6425-6434; Li et al. 2010 Gastroenterology 139, 2158- 2169 e2158; Yu et al. 2013 Cell Stem Cell 13, 328-340). To induce the bifurcation of the bile duct cells, 800 μl of 1.2 mg / ml Type 1 collagen (Corning), 100 μl of 10 × DPBS, 20 μl of 1N NaOH, and 80 μl of water were mixed uniformly on ice Respectively. In addition, 1.2 mg / ml type 1 collagen and 40% metrizol were mixed in a 1: 1 ratio (v / v) to form the cyst form of bile duct tissue. This mixture was mixed with 1 × 10 ⁵ induction liver (1 × 10 ⁵ cells / well) in the same volume of cholangiocyte differentiation medium (CDM: DMEM / F12 mixed with 10% fetal bovine serum (HyClone) and 20 ng / ml HGF Stem cells were mixed with the solution. Subsequently, the cell mixed solution was solidified in a 4-well plate at 37 ° C for 30 minutes. Finally, the media was carefully added onto the prepared gel.

3) periodic-acid staining (PAS) staining

For periodic acid-Schiff (PAS) staining, standard products were used (Periodic Acid-Schiff Kit, Sigma). Cells are fixed in 95% cold ethanol solution supplemented with 10% formalin and washed in running tap water for 1 minute. After the periodic acid solution was treated at room temperature for 5 minutes, it was washed three times with distilled water. Finally, the reaction was carried out with Schiff's reagent solution for 15 minutes at room temperature, followed by washing with tap water for 5 minutes.

4) Indocyanine green (ICG) staining

Indocyanine green (ICG, Sigma) For absorption analysis, ICG solution was added to the culture medium in culture to a final concentration of 1 mg / ml. The cells are then incubated at 37 ° C for 1 hour and then washed three times with PBS solution. Observe whether the ICG is well absorbed into the cells through an optical microscope. After confirming the uptake, the cells were incubated for 6 hours at 37 ° C in normal culture without ICG to confirm the ICG release of the cells.

5) ELISA technique for serum albumin measurement

For the measurement of albumin in the cell culture medium, the cells were cultured for 48 hours. Then, the culture solution was collected and the amount of serum albumin was measured using the Mouse Albumin ELISA Kit (Shibayagi).

6) Analysis of MDR1 receptor activity by Rho123 treatment

Mdr1 (Multidrug resistance protein 1), one of the most important key functions of bile duct cells, one of the receptors related to water and ion release, was measured by the excretion of rhodamine123 in the bile duct cells derived from induced liver stem cells . The bile duct cells were able to transfer Rho123 into the intracellular space, thereby confirming the function of the bile duct cells. Verapamil, an inhibitor of Mdr1, was treated with 10 μM of verapamil (Sigma) 30 minutes before the treatment of Rho123 in order to analyze the inhibitory effect of MDR1 receptor. After washing the cell mixture in the gel state three times with the culture solution, the number of the biliary cell containing Rho123 was observed with a fluorescence microscope.

7) Analysis of CFTR receptor activity by forskolin treatment

The bile duct cells are cultured in a culture medium containing 10 μM calcein-AM (Thermo Fisher Scientific) for 30 minutes. After washing three times with the cell culture medium, the cells are observed under a fluorescence microscope (at 0 hour). In order to observe liquid transport and bile duct cell expansion by Cftr, 10 μM forskolin (FSK, a cAMP agonist; Enzo Life Sciences) and 100 μM IBMX (3-isobutyl-1-methylxanthine, nonselective PDE inhibitor; Sigma) And treated for 24 hours. For inhibition of CFTr activity, 30 μM CFTRinh-172 (a CFTR inhibitor; Sigma) was previously treated 3 hours before FSK / IBMX treatment. After FSK / IBMX treatment, the cells were stained with 10 μM calcein-AM 24 hours later and observed with fluorescence microscope. The total area of the bile duct cells was measured using ImageJ (NIH ver. 1.50), and the degree of dilatation was compared with the size of the non-stimulated bile duct cells.

4. Comparative analysis with existing induction stem cell

In this study, we investigated the effect of differentiation of stem cells on the differentiation of stem cells (Yu B et al., Cell Stem Cell 2013; 13: 328-340) using the transcription factor combination (1b3: Hnf1b, Foxa3) To investigate the effect of E-cadherin or EPCAM positive cells, we used a flow cytometry analyzer for 14 days after transduction. In addition, RNA sequencing analysis, MDR1 receptor activity assay, and qPCR technique were used to compare the characteristics of the inducible stem cell line produced by each combination and the ability to differentiate into bile duct cells. MDR1 receptor activity assay and quantitative PCR method are as described above.

Experiment result

1. Foxa1, Foxa2, Foxa3, Hnf1a and Hnf1b, known as transcription factors essential for hepatic development and liver regeneration, were introduced into the first 50,000 common somatic cells for about 2 weeks, hepatocytes (AFP), bile duct cells (CK7, CK19) It was confirmed that colonies expressing TROP2-specific markers were produced (FIG. 2).

2. Expression of Hnf1b by RT-PCR in order to select the optimal cross-differentiation combination into inducible stem cells. The expression levels of Foxa2, Foxa3 and Hnf1a were significantly lower than those of all five factors. The number of stem cell-specific epithelial cell colonies was significantly reduced when the cells were removed. Based on these results, induction of stem cell induction by induction of Hf1a, Foxa2 (1a2) and Hnf1a and Foxa3 (1a3), respectively, revealed that the number of AFP / CK19 positive colonies and the number of EPCAM- , And it was confirmed that a protein showing liver stem cell characteristics was remarkably expressed in the inducible stem cell produced using 1a3 (Fig. 3a-3f).

3. Induction of stem cell-specific cell lines derived from the combination of 1a3 by RT-PCR, qPCR, and immunofluorescent staining revealed various hepatocyte and bile duct cell and liver stem cell-specific markers (Fig. 4A-4C).

4. In order to confirm the differentiation characteristics of stem cells derived from hepatocyte and biliary stromal stem cells produced by the combination of 1a3, RT-PCR and immunofluorescence staining for 7 days after induction of hepatocyte differentiation in medium containing Matrigel (Ggt, CK7) and liver stem cell marker (Dlk1) were markedly decreased, and hepatocyte-specific functionalities were clearly visible. Cells were able to differentiate into mature hepatocytes under hepatocyte differentiation conditions (Fig. 5A-5E).

5. The differentiation of the cells into collagen and Matrigel-induced differentiation into biliary cells was induced by qPCR, immunofluorescence staining, MDR1 and CFTR receptor activity analysis at 7 days As a result, differentiation into CK19 - expressing bile duct - specific cells on the 3 - dimensional medium was evident. In addition, CBF - specific markers such as CFTr and CK7 were strongly expressed in the body - derived bile duct tissues. In addition, it was shown that cAMP signal transduction was activated during forskolin treatment through the CFTR receptor while absorbing Rho123 via the MDR1 receptor, and thus it was shown to have the ability to differentiate into mature biliary cells under differentiation conditions (Figs. 6A to 6D).

6. In order to compare the induction stem cell crossing differentiation efficiency through the transcription factor combination (1b3) announced by the present inventors to the induction stem cell crossing differentiation efficiency through the combination (1a3) developed by the present inventor, transcription factor introduction As a result of counting E-cadherin or EPCAM positive cells using a flow cytometer, 37.9% versus 11.8% compared to 5.6% and 38.0% versus 11.8%, respectively. Cells were cross-differentiated (Fig. 7).

7. In the case of the inducible stem cell line produced by the combination of 1b3 or 1a3, the inducible stem cell produced by 1a3 is more similar to the derived hepatic stem cell (LEPCs) than the inducible stem cell produced by 1b3 All gene expression patterns were confirmed by RNA sequencing analysis (heatmap and PCA) (Figs. 8a-8b).

8. In vitro demonstration of the ability to differentiate into bile duct cells using the MDR1 receptor activity assay and qPCR technique revealed that the induced stem cell produced by 1a3 absorbed a greater number of Rho123 than the inducible stem cell produced by 1b3 (Fig. 9A-9C). As shown in Fig. 9A-9C, the chimeric cells were differentiated into mature biliary cells, and the biliary cell-specific markers were expressed at a significantly higher level after induction differentiation into biliary cells.

These results suggest that the expression of AFP / CK19 positive colonies, mRNA gene expression pattern, and EPCAM positive cell number were appropriately used after the introduction of the first 5 transcription factors and that Foxa3, a common transcription factor, (1b3) compared with the combination of Hnf1b (1b3) and Hnf1a (1a3), it is possible to cross-differentiate into a highly efficient inducible stem cell. Especially, And the ability to differentiate into bile duct cells can be improved remarkably.

conclusion

In the present invention, in order to improve the low cross-over efficiency and the differentiation efficiency into the bile duct cell of the existing somatic cells, 1) a transcription factor essential for liver development and liver regeneration is strictly selected and finally 1a3 (Hnf1α, Foxa3), 2) cross-differentiation efficiency to high-level inducible stem cells was achieved, and 3) the fundamental characteristics as inducible stem cells and the ability to differentiate into biliary cells in vitro showed a remarkable improvement.

This cross-differentiation study of inducible stem cells is expected to accelerate the development of cell therapeutic agents using inducible stem cells and to validate the toxicity of new drug substances using stem cells derived from stem cells derived from stem cells and to efficiently mass-produce patient-specific differentiated cells And so on.

[Production and Characterization of Induction Bile Duct Stem Cells]

Experimental Method

1. induction of stem cell differentiation, cultivation, establishment of induction bile duct

Induced stem cell lines of the present invention were induced to induce bile duct stromal cells through continuous passaging of about 14 to 16 times in medium containing EGF and HGF (the same composition as HepSCM) (FIG. 11). HepSCM composition / culture conditions were as follows: DMEM / F-12, 10% FBS (Hyclone), 0.1 μM dexamethasone (Sigma), 10 mM nicotinamide (Sigma), 1% ITS (insulin-transferrin-selenium) premix ), penicillin / streptomycin / glutamine (Invitrogen), 10 ng / ml hepatocyte growth factor (HGF; Peprotech), 10 ng / ml epidermal growth factor

2. Characterization of Induction Bile Duct Stem Cells

The induction of stem cells into induced biliary stromal cells was followed up and the relevant signaling system was verified by quantitative PCR (qPCR) and immunofluorescence staining. The experimental method is as described above. Then, 1 x 10 ⁶ induction bile duct embryonic stem cells were transplanted into C57Bl6 / J mouse produced by treating with 0.1% DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) After 4 weeks, differentiation of inducible bile duct embryonic stem cells was performed by immunohistochemistry.

Experiment result

1. Hepatocyte (AFP), biliary cell (CK19), and hepatic stem cell (CK7, TROP2) cells, FoxA3 and Hnf1a, which are essential transcription factors for liver development and liver regeneration, And colonies expressing specific markers were generated (FIG. 12A). (CK19), hepatic stem cells (CK7), and hepatocyte-derived stem cells (CK7) were identified by RT-PCR, qPCR and immunofluorescence staining. TROP2) -specific markers were clearly expressed (Figures 12b-12d).

2. Expression of hepatocyte markers Afp, Alb, and Hnf4a as a result of continuous passage of about 14-16 times in medium containing EGF and HGF (* same composition as HepSCM) (Fig. 13A-13B), while the biliary cell markers such as CK19 and SOX9 were retained. In particular, we confirmed the expression of various markers specific for biliary cells using qPCR, and the expression of markers related to Notch signaling such as Notch2, Hes1, and Jag1 was significantly increased along with the expression of matured biliary cell-specific markers 13c).

These results indicate that the Notch signaling system is deeply involved in the process of inducing stem cell differentiation into inducible biliary stromal cells (1a3-iCPC) through 14 to 16 subculture cultures under the culture conditions of HepSCM used in this study . In order to verify this, 10ng / ml treatment of DAPT, a drug that inhibits Notch signaling, completely suppressed the differentiation into biliary stromal cells and biliary duct cells (maintenance of expression level of Afp, Alb, Ttr, Hnf4a, Fig. 14a-14c) And the state of the stem cells in the induction was maintained rather constantly by qPCR, immunofluorescent staining, and MDR1 receptor activity assay.

3. Inducible biliary stromal cells To verify the ability of the stem cells to differentiate, 1 x 10 ⁶ induction bile duct embryonic stem cells were transplanted into C57B16 / J mice produced by treatment with 0.1% DDC to induce differentiation into biliary ducts, And the degree of differentiation of the transplanted induced biliary stromal cells was analyzed by immunohistochemistry. As a result, a large number of biliary structures were formed in the induction bile duct stromal cells as compared with the induction stem cell (Figs. 15A-15B). Immunostaining with bile duct cell-specific markers such as CK19 and OPN resulted in overwhelming And the induced biliary stromal cells were differentiated into functional biliary duct cells (Figs. 15C-15F).

The results are as follows: 1) the somatic cells are cross-differentiated into the inducible stem cells through the combination of transcription factor 1a3, which is essential for hepatic development and liver regeneration; 2) the cells are continuously passed through the subculture, It is possible to differentiate into biliary stromal cells which are pre-cell stage (see Fig. 1).

conclusion

These results may be useful for the development of cell therapy using biliary stromal cells, modeling of diseases related to cholangiocarcinoma, study of cholangiocellular differentiation and developmental mechanism.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Konkuk University Glocal Industry-Academic Collaboration Foundation <120> Direct conversion method from somatic cells to induced          cholangiocyte stem cells <130> PN170182 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 3203 <212> DNA <213> Mus musculus <400> 1 aaacagagca ggcaggggcc ctgattcact ggccgctggg gccagggttg ggggctgggg 60 gtgcccacag agcttgacta gtgggatttg ggggggcagt gggtgcagcg agcccggtcc 120 gttgactgcc agcctgccgg caggtagaca ccggccgtgg gtgggggagg cggctagctc 180 agtggccttg ggccgcgtgg cctggtggca gcggagccat ggtttctaag ctgagccagc 240 tgcagacgga gctcctggct gccctgctcg agtctggcct gagcaaagag gccctgatcc 300 aggccttggg ggagccaggg ccctacctga tggttggaga gggtcccctg gacaaggggg 360 agtcctgcgg tgggagtcga ggggacctga ccgagttgcc taatggcctt ggagaaacgc 420 gtggctctga agatgacacg gatgacgatg gggaagactt cgcgccaccc attctgaaag 480 agctggagaa cctcagccca gaggaggcag cccaccagaa agccgtggtg gagtcacttc 540 ttcaggagga cccatggcgc gtggcgaaga tggtcaagtc gtacttgcag cagcacaaca 600 tcccccagcg ggaggtggtg gacaccacgg gtctcaacca gtcccacctg tcacagcacc 660 tcaacaaggg cacacccatg aagacacaga agcgggccgc tctgtacacc tggtacgtcc 720 gcaagcagcg agaggtggct cagcaattca cccacgcagg gcagggcgga ctgattgaag 780 agcccacagg cgatgagctg ccaactaaga aggggcgtag gaaccggttc aagtggggcc 840 ccgcatccca gcagatcctg ttccaggcct acgagaggca aaaaaacccc agcaaggaag 900 agcgagagac cttggtggag gagtgtaata gggcggagtg catccagagg ggggtgtcac 960 catcgcaggc ccaggggcta ggctccaacc ttgtcacgga ggtgcgtgtc tacaactggt 1020 ttgccaaccg gcgcaaggag gaagccttcc ggcacaagtt ggccatggac acctataacg 1080 gacctccacc ggggccaggc ccgggccctg cgctgcctgc tcacagttcc cccggcctgc 1140 ccacaaccac cctctctccc agtaaggtcc acggtgtacg gtacggacag tctgcaacca 1200 gtgaggcagc cgaggtgccc tccagcagcg gaggtccctt agtcacagtg tctgcggcct 1260 tacaccaagt atcccccaca ggcctggagc ccagcagcct gctgagcaca gaggccaagc 1320 tggtctcagc cacggggggt cccctgcctc ccgtcagcac cctgacagca ctgcacagct 1380 tggagcagac atctccgggt ctcaaccagc agccgcagaa ccttatcatg gcctcgctac 1440 ctggggtcat gaccatcggg cccggggagc ctgcctccct gggacccacg ttcacgaaca 1500 cgggcgcctc caccctggtt atcggtctgg cctccactca ggcacagagc gtgcctgtca 1560 tcaacagcat ggggagtagc ctgaccacgc tgcagccggt ccagttttcc caaccactgc 1620 atccctccta tcagcagcct ctcatgcccc ccgtacagag ccacgtggcc cagagcccct 1680 tcatggcaac catggcccag ctgcagagcc cccacgcctt atacagccac aagcctgagg 1740 tggcccagta cacgcacacc agcctgctcc cgcagaccat gttgatcaca gacaccaacc 1800 tcagcaccct tgccagcctc acacccacca agcaggtctt cacctcagac acagaggcct 1860 ccagtgagcc cgggcttcac gagccaccct ctccagccac caccatccac atccccagcc 1920 aggacccgtc gaacatccag cacctgcagc ctgctcaccg gctcagcacc agtcccacag 1980 tgtcctccag cagcctggtg ttgtatcaga gttccgactc caacgggcac agccacctgc 2040 tgccatccaa ccatagtgtc atcgagactt ttatctccac ccagatggcc tcctcttccc 2100 agtaaccgtg gtgactgcct cccaggagct gggtccccag ggcctgcact gcctgcatag 2160 ggggtgagga gggccgcagc cacactgcct ggaggatatc tgagcctgcc atgccacctg 2220 acacaggctg ctggccttcc cagaagtcta cgcattcatt gacactgctg ctcctccatc 2280 atcaggaagg gatggctctg aggtgtctca gcctgacaag cgagcctcga ggagctggag 2340 gacggcccaa tctgggcagt attgtggacc accatccctg ctgtttagaa taggaaattt 2400 aatgcttggg acaggagtgg ggaagctcgt ggtgcccgca cccccccagt cagagcctgc 2460 aggccttcaa ggatctgtgc tgagctctga ggccctagat caacacagct gcctgctgcc 2520 tcctgcacct ccccaggcca ttccaccctg caccagagac ccacgtgcct gtttgaggat 2580 taccctcccc accacgggga tttcctaccc agctgttctg ctaggctcgg gagctgaggg 2640 gaagccactc ggggctctcc taggctttcc cctaccaagc catcccttct cccagcccca 2700 ggactgcact tgcaggccat ctgttccctt ggatgtgtct tctgatgcca gcctggcaac 2760 ttgcatccac tagaaaggcc atttcagggc tcgggttgtc atccctgttc cttaggacct 2820 gcaactcatg ccaagaccac accatggaca atccactcct ctgcctgtag gcccctgaca 2880 acttccttcc tgctatgagg gagacctgca gaactcagaa gtcaaggcct gggcagtgtc 2940 tagtggagag ggtaccaaga ccagcagaga gaagccacct aagtggcctg ggggctagca 3000 gccattctga gaaatcctgg gtcccgagca gcccagggaa acacagcaca catgactgtc 3060 tcctcgggcc tactgcaggg aacctggcct tcagccagct cctttgtcat cctggactgt 3120 agcctacggc caaccataag tgagcctgta tgtttattta acttttagta aagtcagtaa 3180 aaagcaaaaa aaaaaaaaaa aaa 3203 <210> 2 <211> 2039 <212> DNA <213> Mus musculus <400> 2 gcgggactcc cgggctgtgt gcctcaggtc ggaactcggg gctagtgcct gtagagagac 60 cgaagcactc ggttccccca ggggggcctc agcctgggtg tgtgggggcg caggccccgg 120 ggatgctggg ctcagtgaag atggaggctc atgacctggc cgagtggagc tactacccgg 180 aggcgggcga ggtgtattct ccagtgaatc ctgtgcccac catggcccct ctcaactcct 240 acatgacctt gaacccactc agctctccct accctcccgg agggcttcag gcctccccac 300 tgcctacagg acccctggca cccccagccc ccactgcgcc cttggggccc accttcccaa 360 gcttgggcac tggtggcagc accggaggca gtgcttccgg gtatgtagcc ccagggcccg 420 ggcttgtaca tggaaaagag atggcaaagg ggtaccggcg gccactggcc cacgccaaac 480 caccatattc ctacatctct ctcataacca tggctattca gcaggctcca ggcaagatgc 540 tgaccctgag tgaaatctac caatggatca tggacctctt cccgtactac cgggagaacc 600 agcaacgttg gcagaactcc atccggcatt cgctgtcctt caatgactgc ttcgtcaagg 660 tggcacgctc cccagacaag ccaggcaaag gctcctactg ggccttgcat cccagctctg 720 ggaacatgtt tgagaacggc tgctatctcc gccggcagaa gcgcttcaag ctggaggaga 780 gggtcagcca 840 cctctgccac cactacagct gccactgcag tcacctcccc ggctcagccc cagcctacgc 900 catctgagcc cgaggcccag agtggggatg atgtgggggg tctggactgc gcctcacctc 960 cttcgtccac accttatttc agcggcctgg agctcccggg ggaactaaag ttggatgcgc 1020 cctataactt caaccaccct ttctctatca acaacctgat gtcagaacag acatcgacac 1080 cttccaaact ggatgtgggg tttgggggct acggggctga gagtggggag cctggagtct 1140 actaccagag cctctattcc cgctctctgc ttaatgcatc ctagcagcgc aattgggaac 1200 gccatgatgg gcgtgggctg caacgttctt gggctctgat ctttctggtt acactttgct 1260 tgtcccatta attaacatct tatttggtct attactgtga tatgacccat tggctactgt 1320 ggtaactgcc atggactctt tggtaggcct agggttgggg tattaggaag gcagatgcgt 1380 ttggaagtgc tgcgaaggtg gtcatgttgg acatattgtg aaggcagtta gactggtgta 1440 ctatgaaagc tgccatatta agtgaagcca ttgggtgatt gatccactgg gtgcctgatg 1500 gtcgtgatgt tggatgacac atgtctggtc ctttggatga tgtgttggac atcttgattg 1560 accttttgag tatgtgacag aacacatctt ctttggctca ttttatcctg ggatcgcctc 1620 ttttttttcc tcttcttttt ctttttcttt ttcttttttt cttttccttt tttctttttt 1680 ttttcttttt tggcagactt cttggttcag cagatgccaa attggccacc atatcacatg 1740 gtgtcttttt tgacattctg gatgcatgga aggtcactgt attggcaagg tgacatctca 1800 gcatgctgct atgcaccaag atagatggtt accacaggcc tgccatcacc atctccttgg 1860 tggaggttgg gtgaggggaa gaggtgagca gaccctatga gttttctctg aagcccatcc 1920 ccaccctgtc tgtgagaaag ggctagtgtg ggtgtcggga gttcctactg aggtcaagtt 1980 cttgtctggg gcttgggaat actgcctgtg tttggccatt aaaaaggcac catctccat 2039

Claims (8)

A method of crossing differentiation into induced cholangiocyte stem cells in somatic cells, comprising introducing Hnf1 alpha and Foxa3 genes into somatic cells of an in vitro isolated mammal.
2. The method of claim 1, wherein the Hnf1 &amp;alpha; gene comprises a nucleotide sequence represented by SEQ ID NO: 1.
2. The method of claim 1, wherein the Foxa3 gene comprises a nucleotide sequence represented by SEQ ID NO: 2.
The method of claim 1, wherein the gene carrier in the gene introduction is a virus, a plasmid, a liposome, or a niozyme.
2. The method according to claim 1, wherein the mammal is a human, a mouse, a rat, a guinea pig, a dog, a cat, a horse, a cow, a pig, a monkey or a chimpanzee.
A composition for crossing differentiation from induced somatic cells into induced cholangiocyte stem cells, comprising a gene carrier for Hnf1? Gene and Foxa3 gene expression.
7. The composition of claim 6, wherein the somatic cell is a fibroblast.
Cross-differentiation into induced cholangiocyte stem cells in somatic cells, including the following steps:
(a) introducing Hnf1 &agr; and Foxa3 genes into somatic cells of mammals isolated from in vitro, to produce inducible stem cells; And
(b) culturing the inducible stem cell of step (a) in a growth factor-containing medium to produce induced biliary stromal cells;

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